Excessive accumulation of extracellular matrix (ECM) proteins is the distinctive feature of fibrotic skin conditions such as hypertrophic scars keloids and localized or systemic sclerosis (scleroderma). This process is dependent on activation of the synthesis of ECM in interstitial fibroblasts, which are often transformed to myofibroblasts that are positive for smooth muscle α-actin (alpha smooth-muscle actin, α-SMA), a marker that is indicative of differentiation to cells with a high rate of proliferation and production of extracellular matrix (Jiménez et al., 1996; Jelaska and Korn, 2000).
One of the key molecular factors in fibrotic processes is transforming growth factor β (TGF-β), which is overexpressed in the majority of fibrotic diseases and displays various profibrotic effects in fibroblasts (Querfeld et al., 1999; Chiller et al., 2004). Activation of the TGF-β receptors leads to the activation of various kinase signalling cascades, leading to phosphorylation of SMAD proteins, as well as to the activation of SMAD-independent kinases, which together activate the synthesis of ECM and the growth of fibroblasts and differentiation to give rise to myofibroblasts (Shi and Massague, 2003; Daniels et al., 2004).
Connective-tissue growth factor (CTGF), for its part, is a soluble mediator that is induced rapidly and selectively in the fibroblasts by the action of TGF-β (Leask et al., 2004). CTGF has also been detected specifically in fibrotic skin diseases (Igarashi et al., 1996) and, in animal models, promotes and perpetuates the profibrotic effects of TGF-β (Frazier et al., 1996). Although the role of epidermal CTGF in fibrosis has not been well established, earlier studies show that it is expressed by normal keratinocytes in vivo (Quan et al. 2002) and that the negative regulation of its levels caused by ultraviolet radiation appears to be linked to reduction in the synthesis of procollagen induced by said radiation (Quan et al., 2002).
Although the majority of fibrotic diseases usually start with variable degrees of inflammation, anti-inflammatory treatments are not effective when used in the treatment of chronic fibrotic diseases, which represent an important group of lesions for which there is no specific treatment. TGF-β appears to be an attractive target for the treatment of fibrotic diseases; in fact, various anti-TGF-β strategies have been tried successfully in animal models for fibrosis, including various murine models of scleroderma (MacCormick et al., 1999; Yamamoto et al., 1999b; Zhang et al., 2003; Lakos et al., 2004). In the animal model of bleomycin-induced cutaneous sclerosis (a model that reproduces most of the characteristics of human scleroderma, such as infiltration of the skin with inflammatory cells, vascular damage, activation of mast cells and prolonged skin fibrosis (Yamamoto et al., 1999c), previous studies showed that both the administration of anti-TGF-β antibodies and genetic deficiency of SMAD3 inhibit the development of fibrosis, which lends strong support for a fundamental role of TGF-β (Yamamoto et al., 1999b; Lakos et al., 2004) in the genesis and development of sclerosis. In studies using the animal model of bleomycin-induced scleroderma, systemic treatment with anti-TGF-β antibodies reduces fibrosis in parallel with a reduction in mast cells and in infiltration of inflammatory cells (Yamamoto et al., 1999b). The relevance of the mast cells in the models of skin fibrosis is uncertain, because earlier studies in mastocyte-deficient mice demonstrated that their contribution to the development of fibrosis is not indispensable (Everett et al., 1995; Yamamoto et al., 2001). For its part, infiltration of inflammatory cells plays an important role in the early stages of development of fibrosis, but its role is less clear in the later stages, in which it may disappear or persist independently of the progression of the fibrosis, but usually fibrosis progresses in the absence of significant infiltration of inflammatory cells.
Despite these encouraging results, inhibition of TGF-β by the systemic route is a cause of disquiet regarding its safety, because this factor triggers powerful pleiotropic effects in immunomodulation, inflammation and in the development of tumours (Akhurst, 2002). In keeping with this, in mice deficient in TGF-β1, the formation of scars on the skin was reduced, but the animals developed cachectic syndrome (characterized by pronounced weight loss), accompanied by a generalized inflammatory response and tissue necrosis, resulting in organ failure and death (Bottinger et al., 1997). Therefore local instead of systemic inhibition of TGF-β represents an alternative strategy for the development of antifibrotic treatments (Daniels et al., 2004; Lakos et al., 2004). Local inhibition of TGF-β had been tried before, by direct application of neutralizing antibodies on open wounds on the skin or the cornea, but the application of antibodies or of large peptides with the intention that they should cross the epidermal barrier was not found to be a very practical strategy (Jester et al., 1997; Brahmatewari et al., 2000), as these molecules are too large and their diffusion through the epidermal barrier is hampered.
It would be interesting to find a compound with sufficient capacity to diffuse through the epidermal barrier, capable of inhibiting TGF-β when administered locally, and with a capacity for curing or ameliorating the effects of skin fibrosis in mammals. Moreover, if said molecule is really to be useful for the treatment of humans, it would be necessary to develop a pharmaceutical form suitable for topical administration of said active compound, which not only would permit local action of the compound without being absorbed significantly, but would display good spreading capacity and a pleasant appearance without being greasy.
These requirements are met by the novel use of peptides, including peptides that are already known, including in particular the peptide designated P144, whose use is described in the present invention for the treatment of skin fibrosis by its topical application, compositions also being supplied which contain said peptide and are suitable for topical administration for humans. Peptide P144, described in international patent application WO 00/31135, is an antagonist of TGF-β1 which comprises the amino acids 730 to 743 of the type III receptor (β-glycan) of human TGF-β1 (accession number Q03167, SwissProt) and had been demonstrated in vitro to be capable of interfering with the binding of TGF-β1 to its cellular receptors in Mv-1-Lu cells and preventing the inhibition of proliferation of said cells induced by TGF-β1. Conversely, when administered intraperitoneally it gives rise to a powerful antifibrotic response in the liver of rats, in which liver cirrhosis is induced by inhalation of carbon tetrachloride. However, its topical administration, as well as its possible usefulness in the treatment of established skin fibrosis, had not heretofore been described. Compositions suitable for the topical administration of said peptide had not been described either; this need is also covered by the present invention.